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研究生： 陳柏亨
Chen, Po-Heng
論文名稱： 透過濕式靜電紡絲生產三維高分子奈米纖維支架與評估其對裝載牙髓幹細胞後之增生與分化效果
Evaluation of the Proliferative and Differentiative Effects of a Three-Dimensional Polymeric Nanofiber Scaffold Produced by Wet Electrospinning on Post-Pulp Stem Cells
指導教授： 吳炳慶
Wu, Ping-Ching
學位類別： 碩士
Master
系所名稱： 工學院 - 生物醫學工程學系
Department of BioMedical Engineering
論文出版年： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 80
中文關鍵詞： 濕式靜電紡絲 、聚乳酸-甘醇酸 、聚乙烯醇 、奈米纖維支架 、奈米顆粒 、藥物緩釋 、膠原蛋白 、地塞米松 、細胞外基質 、牙髓幹細胞 、細胞分化 、牙本質再生
外文關鍵詞： Wet electrospinning, Polylactic acid-glycolic acid, Polyvinyl alcohol, Nanofiber scaffold, Nanoparticles, Drug release, Collagen, Dexamethasone, Extracellular matrix, Dental pulp stem cells, Cell differentiation, Dentin regeneration
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    • 全世界約有24.3億人有恆牙齲齒/腐爛的問題，佔總人口的36％，其中兒童患者有6.2億(佔總人口的9％）。此外，年齡在20到64歲之間的美國人中有92％的人有齲齒現象，幾乎四分之一的年齡組有未經治療的蛀牙。如果不進行治療，感染可能會擴散到周圍組織導致牙齦壞死。嚴重情況下，細菌隨血液留至心臟瓣膜上落地生根，導致感染性心內膜炎。由此可見牙齒對人們健康與生活品質上具有極大影響力的器官，因齲齒、外傷、牙周疾病或基因缺陷等症狀，造成牙齒缺失的疾病，使病人在咀嚼、發音等生理狀態或外型有所困擾。
    現今醫療上治療齲齒與缺牙患者一般是透過補牙和植牙方式治療，嚴重者則需要根管治療。儘管牙齒植體的材料與相關技術日新月異，但長時間填補材料所釋放出的殘留物會造成細胞毒性及基因毒性，影響到正常牙細胞生長，若能發展出具修復齲齒的牙髓幹細胞材料支架，再患者自身的齲齒部位再生牙本質組織，應是完美的治療方式，亦將是臨床治療的新突破。
    至今許多文獻已證明牙髓幹細胞是一種多潛能性幹細胞(multipotent)，有分化為牙本質母細胞 (odontoblasts)和促進牙本質再生(dentinogenesis)的特性，可被應用於治療齲齒及覆髓(capping)細胞治療(cell therapy)潛力。我們利用濕式靜電紡絲的技術，製作出蓬鬆三維多孔隙之聚乙烯醇(PVA)奈米纖維材料，基於電紡後奈米纖維的疊合，形成高密度填充孔隙的3D支架，模擬體內細胞與細胞之間細胞外基質的微環境，輔助牙髓幹細胞生長的載體。此外，將含有地塞米松化合物的PLGA奈米顆粒混入奈米纖維材料中，以及將奈米纖維鍍上I型膠原蛋白，形成地塞米松@PLGA /PVA/膠原蛋白奈米纖維複合物支架。研究結果顯示將牙髓幹細胞培養於地塞米松@PLGA /PVA/膠原蛋白奈米纖維複合物支架中，培養3天，與2D環境中培養牙髓幹細胞之細胞活性比較下，透過地塞米松@PLGA /PVA /膠原蛋白奈米纖維複合物支架培養牙髓幹細胞的細胞活性提高122%。因為地塞米松@PLGA /PVA /膠原蛋白奈米纖維能模擬細胞外基質3D微環境特性，促使牙髓幹細胞於材料上貼附及生長增生。另一方面，PLGA奈米顆粒能緩釋放出地塞米松的特性，進一步誘導牙髓幹細胞產生牙本質再生。地塞米松@PLGA奈米顆粒混入奈米纖維中，可以將纖維與纖維間的空隙撐開，藉此來製造寬的孔隙，有利於細胞向內增生。另一方面，於地塞米松@PLGA /PVA靜電紡絲奈米纖維鍍上I型膠原蛋白，可模擬牙齒結構中釉柱及膠原蛋白柱緊密疊合的構型，助於細胞貼附生長。
    特別是牙本質牙髓複合體的再生，將其重點放在自身牙齒器官的修復，將會比傳統牙髓加蓋療法更有效可靠和更安全的方法。我們研究之地塞米松@PLGA /PVA/膠原蛋白複合型奈米纖維支架，於未來應用在牙髓腔牙本質再生醫療上，具潛力之高分子生物性3D支架材料。

    There are approximately 2.43 billion people (36% of the world's population) with decayed or decayed permanent teeth, of which 620 million are children. (9% of the total population). In addition, 92% of Americans between the ages of 20 and 64 have dental caries, and nearly a quarter of the age group have untreated tooth decay. In recent years medical techniques have used reinforced resin fillers such as padding, but over time the filler can easily fall off, causing inflammation or bacterial infection. If left untreated, the infection may spread to surrounding tissues leading to gingival necrosis. In severe cases, bacteria remain in the blood of the heart valve root, resulting in the infection of the endocarditis. Therefore, teeth have a significant impact on the health of people and their quality of life. Due to symptoms such as dental caries, trauma, periodontal disease, genetic defects, the disease causes some problems of tooth loss, causing the patient to chew, pronounce or have other physiological states or appearances.
    Today's medical treatment of dental caries and edentulous patients is generally treated by filling and implanting; in severe cases, root canal treatment is needed. Despite the new materials and the associated development of dental implants, the residues released by the materials over a long time can cause cytotoxicity and genotoxicity, affecting the growth of normal dental cells, and if the dental pulp stem cell material scaffold with dental caries is developed, the regeneration of the dental tissue in the patient's own caries should be a perfect treatment and a new breakthrough in the treatment of the trampoline.
    In recent years, many papers have shown that dental pulp stem cells are a multi-potent, which differentiate into odontoblasts and promote dentinogenesis. It can be used to treat dental caries and cap cell therapy potential. We use wet electrospinning technology to produce loose three-dimensional porous polyvinyl alcohol (PVA) nanofiber material as a cell-attached growth scaffold based on the superposition of electrospun nanofibers, a high-density filled pore 3D scaffold of the pores simulates the microenvironment of extracellular matrix (ECM) between cells in the body and supports the growth of dental pulp stem cells. Additionally, PLGA nanoparticles containing dexamethasone compounds were mixed into the nanofiber material , and the nanofibers were coated with type I collagen to form a Dexamethasone @PLGA / PVA / collagen nanofiber composite scaffold. Our research results show that dental pulp stem cells were cultured in Dexamethasone @PLGA /PVA /Collagen nanofiber composite scaffold for 3 days. Compared with the cell viability of dental pulp stem cells cultured in a 2D environment, the cell viability of dental pulp stem cells cultured through the Dexamethasone @PLGA /PVA /Collagen nanofiber composite scaffold increased by 122%. Because Dexamethasone @PLGA/ PVA /Collagen nanofibers can simulate the ECM 3D micro -environmental properties, and promote the attachment and growth of dental pulp stem cells to materials. On the other hand, PLGA nanoparticles can slowly release the properties of dexamethasone compounds and further induce dentin regeneration in dental pulp stem cells. By mixing the Dexamethasone @nanoparticle into the nanofiber, the gap between the fiber and the fiber can be opened, thereby creating a wide pore, which is favorable for the cell to inwardly proliferate. Furthermore, Dexamethasone @PLGA/ PVA electrospinning nanofibers are coated with type I collagen that can simulate the closely overlapping configuration of enamel rod and collagen rod in the tooth structure, which can facilitate dental pulp stem cells attachment and growth.
    In particular, the regeneration of the dentin-pulp complex, focusing on the repair of its own dental organs, will be more effective, reliable and safer than traditional endodontic capping therapy. We developed the Dexamethasone @PLGA/ PVA/ collagen composite nanofiber scaffold, to future applications in regenerative medicine dentin pulp cavity, with the potential of the 3D molecular biological scaffold.

    摘要 I Abstract III 誌謝 VI Table of contents VIII List of figures XI Abbreviation index XIII Chapter 1. Introduction 1 1.1 The number of cases in the United States 2 1.1.1 Clinical disease treatment and risk 2 1.1.2 Untreated dental disease problems 2 1.1.3 Clinical status and challenges 2 1.2 Tissue engineering and biological scaffolds 3 1.2.1 Electrospinning 4 1.2.2 Electrospinning technology applied to biological scaffolds 4 1.2.3 Simulating the importance of Extracellular Matrix (ECM) 6 1.3 Biomaterial Degradation Characteristics 6 1.4 Drug Delivery of Poly(lactic-co-glycolic acid) (PLGA) nanoparticles 7 1.5 Electrospinning of Polyvinyl alcohol (PVA) Characteristics 8 1.6 Type I collagen applied to nanofiber technology 8 1.7 Dental stem cell types 8 1.7.1 Reasons for the decision to use dental pulp stem cells 9 1.7.2 Dental pulp stem cell surface antigen markers 10 1.7.3 Compounds that induce the differentiation of pulp stem cells 10 1.8 Hypothesis and Specific Aims 11 Chapter 2. Materials and Methods 12 2.1 Materials 13 2.2 The establishment of an electrospinning machine 14 2.2.1 Electrospinning machine 14 2.2.2 Wet-Electrospinning machine 15 2.3 Electrospinning process 16 2.3.1 Electrospinning PVA nanofibers 16 2.3.2 Wet-Electrospinning PVA nanofibers 16 2.3.3 Wet-Electrospinning PLGA(nanoparticle)/ PVA nanofibers 17 2.4 Preparation of nanoparticles 17 2.4.1 Synthesis of 12nm FePt nanoparticles 17 2.4.2 Synthesis of Dexamethasone-loaded PLGA nanoparticles 18 2.4.3 Synthesis of FePt-loaded PLGA nanoparticles 18 2.5 Fabrication of nanofibers scaffolds 19 2.5.1 Fabrication of 2D FePt@PLGA/ PVA scaffolds 19 2.5.2 Fabrication of 3D Dexamethasone @PLGA/ PVA scaffolds 19 2.5.3 3D Nanofibers scaffolds for Dip collagen using a Dip coating 20 2.6 Characterization of nanofibers scaffolds 21 2.6.1 Scanning electron microscopy (SEM) of scaffolds 21 2.6.2 Transmission electron microscopy (TEM) of scaffolds 21 2.6.3 2D&3D Nanofibers porosity characterization 21 2.6.4 Fourier transformer infrared spectrophotometric analysis (FTIR) for coating collagen nanofibers 22 2.6.5 Dexamethasone release form Dexamethasone @PLGA/ PVA nanofibers 22 2.7 Mechanical properties of PVA & PLGA(Nanoparticles)/ PVA nanofibers tensile testing and resilience testing 22 2.8 In vitro biodegradation testing 23 2.9 Nanofiber contact angle testing 23 2.10 Cell Culture 23 2.10.1 Dental pulp stem cells (DPSCs) in primary cell culture 23 2.10.2 Cell culture of 4T1 Cells 24 2.10.3 The cell culture of NIH-3T3 cells 24 2.10.4 Cell culture of DPSCs with block 3D nanofiber scaffolds on 24 plates 25 2.10.5 Cells culture of DPSCs with thin 3D nanofiber scaffolds on coverslips 25 2.10.6 Cell counts 26 2.11 PLGA(Nanoparticles)/ PVA/ Collagen Nanofibers cytotoxicity assay 26 2.12 Colony formation assay of DPSCs in vitro 27 2.13 Immunofluorescence for Identifying the DPSCs 27 2.14 The Flow Cytometry assay of DPSCs 28 2.15 DPSCs Osteogenic Differentiation 28 2.16 DPSCs Osteogenic Mineralization Assay 28 2.17 Fluorescence image quantization 29 2.18 Statistical Analysis 29 Chapter 3. Results 30 3.1 Characterization of electrospinning nanofibers 31 3.1.1 Morphology and pore size of PVA & Wet-PVA Nanofibers 31 3.1.2 Morphology of PLGA(Nanoparticles)/ PVA nanofibers by wet electrospinning 31 3.2 Mechanical properties of Wet-PLGA(Nanoparticles)/ PVA Nanofibers scaffold 32 3.3 Cytotoxicity Analysis of Wet-PLGA(Nanoparticles)-PVA/Collagen Nanofibers 32 3.4 Dip coating Collagen type I of Wet- PVA nanofibers 32 3.5 Contact angle test of Wet-PLGA(Nanoparticles)/ PVA Nanofibers 33 3.6 Biodegradability of Wet-PLGA(Nanoparticles)/ PVA Nanofibers 34 3.7 Identification of primary culture dental pulp stem cells 34 3.7.1 Morphology of dental pulp stem cells 34 3.7.2 Colony formation assay of DPSCs in vitro 35 3.7.3 Flow Cytometry of DPSCs in vitro 35 3.7.4 Fluorescent analysis of DPSCs in vitro 35 3.7.5 Osteogenic Differentiation of DPSCs 35 3.8 Wet-PLGA(Nanoparticle)/ PVA Nanofibers scaffold influence the proliferation of DPSCs 36 3.9 The Cell Viability of the Wet-PLGA(Nanoparticle)/ PVA/ Collagen Nanofibers scaffold 36 3.10 Characteristics of the Dexamethasone@ PLGA Nanoparticles 37 3.11 Drug delivery of Dexamethasone @PLGA/ PVA Nanofibers scaffold in vitro 37 3.12 Drug delivery of Dexamethasone @PLGA/ PVA/ Collagen Nanofibers scaffold differentiation of DPSCs 38 Chapter 4. Discussion 40 4.1 To Proof the function of PLGA/ PVA/ Nanofibers 41 4.2 The cells are grown on coverslips containing nanofibers 42 4.3 The effects of electrospinning porosity on cell growth 44 Chapter 5. Conclusion 45 5. Conclusion 46 Tables and Figures 47 References 80

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